De multiples micro-organismes ont la capacité de catalyser l’oxydation électrochimique de matières organiques en s’organisant en biofilm à la surface d’anodes. Ce processus est à la base de procédés électro-microbiens très innovants tels que les piles à combustible microbiennes ou les électrolyseurs microbiens. L’interface biofilm/électrode a été l’objet de nombreuses étudesdont les conclusions restent difficiles à démêler en partie du fait de la diversité des paramètres interfaciaux mis en jeu. L’objet de ce travail de thèse est d’exploiter les technologies microsystèmes pour focaliser l’impact de la topographie de surface des électrodes sur le développement du biofilm et sur ses performances électro-catalytiques. La formation de biofilmsélectroactifs de Geobacter sulfurreducens a été étudiée sur des électrodes d’or présentant des topographies bien contrôlées, sous la forme de rugosité, porosité, réseau de piliers, à des échellesallant du nanomètre à quelques centaines de micromètres. La présence de microrugosité a permis d’accroitre les densités de courant d’un facteur 8 par rapport à une surface lisse et son effet a étéquantifié à l’aide du paramètre Sa. Nous avons tenté de distinguer les effets des différentes échelles de rugosité sur le développement du biofilm et la vitesse des transferts électroniques.L’intérêt de la microporosité a été discuté. L’accroissement de surface active par la présence de micro-piliers s’est avéré très efficace et une approche théorique a donné des clés de compréhension et d’optimisation. Les connaissances acquises dans les conditions de culture pure ont finalement été confrontées avec la mise en oeuvre de biofilms multi-espèces issus d’un inoculum complexe provenant de sédiments marins.

In nature, biofilm is a complex structure resulted of multicellular bacterial communities that provide important nutritional functions and the acquisition of protective traits such as antibiotics resistance and horizontal gene transfer. The development from the planktonic, lonely bacteria, to the mature multilayered biofilm structure consists of three main phases: motility, attachment and biofilm maturation. At cellular level, the process is controlled by several genes such as flhD, fliA, rpoS, csgD, adrA, cpxR all acting as master regulators. Additionally, the global regulators CRP, IHF, Fis, and others in less frequency, have been related to biofilm formation, although blurry information has been provided. In this thesis we used synthetic, molecular and cellular biology approaches to understand the effect of CRP, IHF and Fis in the transcriptional regulatory network in the bacterium Escherichia coli. In the first chapter, we employed network analysis to reconstruct and analyze part of the entire regulatory network described to modulate the flagella-biofilm program. With this analysis we identified some critical interactions responsible for the planktonic-biofilm transition. Next, we selected the top ten effectors nodes of the network and cloned the promoter region of those genes in a reporter system. As extensively explained in chapter II, this system allowed us to validate as well as suggest new interactions in the network. Additionally, the measurement of the promoter activity during bacterial development show that CRP, IHF and Fis differentially modulate most of the surveyed genes suggesting that those Global Regulators participate to modulate gene expression in different phases of the planktonic-biofilm development. At chapter three, to get a better overview of the entire process, we performed motility, adherence/early biofilm and mature biofilm assays. We describe the intrinsic ability of E. coli to perform motility, adherence and mature biofilm at 37?C. In contrast, the absence of ihf, fis as well as Carbon Catabolite Repression (CCR), lead to altered phenotypes at both motility and biofilm development. At the end, we discussed how the changes of promoter activity of target genes, together with our network analysis, could explain part of the altered phenotypes observed. For instance, we observed changes at the main stress responders rpoS and rpoE that, in combination with alterations at specific genes such as fliA, can explain the enhanced motility in the E. coli ?ihf strain. Altogether, in this thesis, we provided evidence that CRP, IHF and Fis control the activity of the promoter regions of genes involved in the planktonic-biofilm development. / Na natureza, o biofilme é uma estrutura complexa resultante de comunidades bacterianas multicelulares que fornece importantes funções nutricionais e a aquisição de traços de proteção como resistência a antibióticos e transferência horizontal de genes. O desenvolvimento das bactérias planctônicas solitárias para uma estrutura de biofilme maduro consiste em três fases principais: motilidade, fixação e maturação do biofilme. Ao nível celular, o processo é controlado por vários genes tais como flhD, fliA, rpoS, csgD, adrA, cpxR, todos agindo como reguladores mestre. Além disso, os reguladores globais CRP, IHF, Fis e outros em menor freqüência, têm sido relacionados à formação de biofilme, embora tenham sido fornecidas informações nao conclusivas sobre esse processo. Nesta tese foram utilizadas abordagens de bioinformática, assim como de biologia molecular e celular para entender o efeito de CRP, IHF e Fis na rede reguladora da transição de motilidade para biofilme na bactéria Escherichia coli. No primeiro capítulo, utilizamos a análise de rede para reconstruir e analisar parte da rede regulatória descrita para modular o programa flagelo-biofilme. Com esta análise identificamos algumas interações críticas responsáveis pela transição planctônica-biofilme. Em seguida, selecionamos os dez principais nós efetores da rede e clonamos a região promotora desses genes em um sistema repórter. Conforme explicado amplamente no capítulo II, este sistema nos permitiu validar e sugerir novas interações na rede. Adicionalmente, a medição da atividade do promotor durante o desenvolvimento bacteriano mostra que a CRP, a IHF e a Fis modulam diferencialmente a maioria dos genes analisados sugerindo que estes Reguladores Globais participam para modular a expressão génica em diferentes fases do desenvolvimento de estado planctónico para biofilme. No capítulo três, para obter uma melhor visão geral de todo o processo, realizamos ensaios de motilidade, aderência / biofilme precoce e biofilmes maduros. Descrevemos a capacidade intrínseca de E. coli para realizar motilidade, adesão e biofilme maduro a 37 °C. Em contraste, a ausência de ihf, fis, bem como o fenômeno de Repressão de Catabolite de Carbono (CCR), levam a fenótipos alterados, tanto na motilidade como no desenvolvimento do biofilme. No final, discutimos como as mudanças da atividade do promotor de genes alvo, juntamente com a nossa análise de rede, poderia !xi explicar parte dos fenótipos alterados observados. Por exemplo, observamos mudanças nos principais respondedores de estresse rpoS e rpoE que, em combinação com alterações em genes específicos como fliA, podem explicar a motilidade aumentada na estirpe de E. coli ?ihf. Em conjunto, nesta tese, apresentamos evidências de que CRP, IHF e Fis controlam a atividade das regiões promotoras de genes envolvidos no desenvolvimento planctônico-biofilme.

Hipersensibilidade dentária (HD) é definida como uma dor decorrente de dentina exposta em resposta a fatores intra-bucais. Porém, o papel do biofilme supragengival (BS) no processo de desencadeamento e/ou aumento da HD não é totalmente esclarecido. Este estudo é um ensaio clínico de braço único cujo objetivo foi o de avaliar se, após 4 dias sem medidas mecânicas de controle do BS, a auto-percepção da hipersensibilidade dentinária é influenciada pela presença daquele. Para isto, 74 participantes (28,1±7,3 anos, 48 mulheres), com saúde gengival e ≥ 5 dentes hígidos por quadrante, foram incluídos se apresentassem recessão (REC) e/ou queixa de HD. No dia 0, avaliou-se REC, escala Schiff (ES), Escala Visual Analógica, sensibilidade tátil por meio da sonda Jay (JAY) e Índice de Sangramento Gengival (ISG). Após, realizou-se profilaxia, para remoção de cálculo e/ou BS e os participantes foram orientados a abster-se do controle mecânico do BS por 4 dias e a realizar 2 bochechos/dia (manhã e noite, 1 minuto) com solução de creme dental fluoretado (1g/9ml). No dia 4, após exame usando o Índice de Placa Visível (IPV), índice de Quigley-Hein (QH), EVA e ISG, foi realizada nova profilaxia. Regressão logística por meio de Equações de Estimação Generalizadas foi conduzida, sendo o dente a unidade de análise. Uma média de 6,00±4,08 dentes/paciente apresentou recessão e/ou queixa de HD. Na totalidade dos dentes (n=444), o acúmulo do BS não determinou aumento na queixa de HD. Quando analisados dentes com Schiff≥1 no dia 0 (n=128), a presença de BS associou-se ao aumento na EVA (OR=4,233; p=0,006) independente da presença ou extensão da REC. Conclui-se que o acúmulo do BS aumenta a auto-percepção de HD. / Dental hypersensitivity (HD) is defined as a pain arising from exposed dentin in response to intra-buccal factors. However, the role of the supragingival biofilm (BS) in the process of triggering and / or increasing HD is not fully understood. This study is a single-arm clinical trial whose objective was to evaluate whether, after 4 days without mechanical measures of BS control, the self-perception of dentin hypersensitivity is influenced by the presence of that. Seventy-four participants (28.1 ± 7.3 years, 48 women) with gingival health and ≥ 5 healthy teeth per quadrant were included if they presented recession (REC) and / or HD complaint. At day 0, REC, Schiff scale (ES), Visual Analog Scale tactile sensitivity by means of the Jay (JAY) probe and Gingival Bleeding Index (GBI) were performed. After that, prophylaxis was performed, for calculation and / or BS removal, and participants were instructed to abstain from BS mechanical control for 4 days and to perform 2 mouthwashes / day (morning and night, 1 minute) with a fluoride toothpaste slurry (1g / 9ml). On day 4, after examination using the Visible Plaque Index (IPV), Quigley-Hein Index (QH), EVA and GBI, a new prophylaxis was performed. Logistic regression using Generalized Estimation Equations was conducted, with the tooth being the unit of analysis. An average of 6.00 ± 4.08 teeth/patient presented recession and / or complaint of HD. In all teeth (n = 444), the accumulation of BS did not increase HD complaint. When teeth with Schiff≥1 on day 0 were (n = 128), the presence of BS was associated with an increase in VAS (OR = 4,233; p = 0.006) regardless of the presence or extent of REC. It is concluded that the accumulation of BS increases the self-perception of HD

The historical notion of a microbial population has been of a clonal population of identical swimming planktonic cells in a laboratory flask. As the field has advanced, we have grown to appreciate the immense diversity in microbial behaviors, from their propensity to grow in dense surface-attached communities as a biofilm, to the consequences of social dilemmas between cells, to their ability to form spores able to survive nearly any environmental insult. However, the historically biased view of the clonal microbial population still persists – even when a rare phenotype is investigated, the focus simply shifts to that narrower focal population - and this bias can lead to some of the broader questions relating to the consequences of phenotypic diversity within populations to be overlooked. This work seeks to address this gap by investigating the evolutionary causes and consequences of phenotypic heterogeneity, with a focus on clinically relevant phenotypes. We first develop and experimentally validate a theoretical model describing the evolution of a microbial population faced with a trade-off between survival and fecundity phenotypes (e.g. biofilm and planktonic cells), which suggests that simultaneous investment in both types maximizes lineage fitness in heterogeneous environments. This model helps to inform the experimental studies in the following chapters. We find that biofilm-mediated phenotypic resistance to antibiotics is evolutionarily labile, and responsive to antibiotic dose and whether biofilm or planktonic cells are passaged. We also show that persistence in E. coli is age-independent, supporting the current hypothesis of stochastic metabolic fluctuations as the cause of this rare phenotype. Finally, we explore phenotypic variation across a library of natural isolates of P. aeruginosa, and find few organizing principles among key phenotypes related to virulence. Together these results suggest that phenotypic heterogeneity is a crucial component in the ecology and evolution of microbial populations, and directly affects pressing applied concerns such as the antibiotic resistance crisis.

A água do equipo odontológico pode servir como meio de disseminação de microrganismos, uma vez que é a segunda maior fonte de contaminação na Odontologia. O objetivo desta pesquisa foi avaliar o nível de contaminação por bactérias aeróbias totais em água de equipos odontolóicos (reservatórios, seringas tríplices e alta rotação) e torneiras de 5 Clínicas Odontológicas da Faculdade de Odontologia de Ribeirão Preto - USP, por meio do método convencional (R2A Agar) e o sistema Petrifilm AC (3M St, Paul, MN, USA). Além disso, determinou-se o nível de contaminação por Pseudomonas spp. (Cetrimide Agar Base), coliformes (Endo Agar) e fungos (Petrifilm YM para bolores e leveduras), como também identificou-se as bactérias com série bioquimica na forma de kit comemial (API 20NE), detectou-se bactérias da boca pela técnica de Checkerboard DNA-DNA Hybridization e analisou-se o biofilme formado nas linhas dágua dos equipos (seringas tríplice e alta rotação), com auxilio de microscópio eletrônico de varredura (MEV). Por outro lado, foram sugeridas recomendações para a manutenção da qualidade microbiológica da água de equipos odontológicos. As comparações estatísticas mostraram que os níveis de contaminação bacteriana das águas de torneiras, bem como dos equipos foram mais elevados pelo método R2A Agar do que pelo sistema Petrifilm AC (p<0,001). Embora, as amostras de água das torneiras utilizadas para preencher os reservatórios dos equipos tivessem pequeno número de bactérias (908UFC/ml) - R2A e (2UFC/ml) Petrifilm AC, as dos 25 equipos apresentaram: reservatórios de 0 a 3.900.000 UFC/ml (média de 211.705 UFC/ml) - R2A Agar, e de 0 a 231.000 UFC/ml (média de 14.065 UFC/ml) Petrifilm AC; seringas tríplices de 0 a 5.200.000 UFC/ml (média de 509.068 UFC/ml) - R2A Agar, e de 0 a 610.000 UFC/ml (média de 30.842 UFC/ml) Petrifilm AC; e alta rotação de 0 a 6.300.000 UFC/ml (média de 862.279 UFC/ml) - R2A Agar, e de 0 a 730.000 UFC/ml (média de 61.817 UFC/ml) Petrifilm AC. As placas Petrifllm AC incubadas a 23°C por 7 dias demonstraram um nível maior de contaminação bacteriana do que aquelas incubadas a 35°C por 48h (p<0,001). De acordo com o método de cultura, Escherichia coli, coliformes totais e Pseudomonas spp. estavam ausentes das amostras de água das torneiras, embora 11 (44%) dos equipos tivessem apresentado E. coli e/ou coliformes totais e em 1 (4%) de alta rotação, Pseudomonas aeruginosa. Todavia, segundo o método molecular, 1 (50%) amostra de água de torneira e 36 (48%) de equipos mostraram contaminação por E. coli. Das águas de 10 torneiras e 25 equipos, 1 (10%) e 17 (68%) estavam contaminadas com fungos, respectivamente. As análises com MEV mostraram biofilmes nas linhas d\'água de todos os equipos, constituídos por uma diversidade microbiana embutida em densas e extensas matrizes de substâncias poliméricas extracelulares. As bactérias identificadas por meio do API 20 NE foram Acinetobacter Iwoffii, Brevundimonas vesicularis, Burkholderia cepacia, Moraxella spp., Oligel/a ureo/ytica, Pasteurella spp., P. aeruginosa e Sphingomonas paucimobi/is. Nas águas dos equipos, as bactérias mais prevalentes exclusivas da boca forem Streptococcus gordonii (35/46,7%), Treponema denticola (28/37,3%), e Aggregatibacter actinomycetemcomitans(b) (9/25,6%). Em conclusão, o BIOFILME formado nas linhas d\'água dos equipos funciona como um \"sistema amplificador\" do pequeno número de microrganismos das águas de torneiras, sendo a causa principal dessa alarmante contaminação das águas dos equipos. Assim, recomendações para a manutenção da qualidade microbiológica da água de equipos, bem como a avaliação de fungos deveriam ser acatadas pelos profissionais da Odontologia. / Dental unit water may serve as microorganism dissemination, since it is the second major source of contamination in dentistry. The aim of this research was to assess the contamination level of total aerobic bacteria in water from dental units (reservoirs, air-water syringes and high-speed handpieces) and taps from 5 Dental Clinics at the Faculdade de odontologia de Ribeirão PReto USP using conventional method (R2A Agar) and Petrifilm SYSTEM (3m, St Paul, MN, USA). Moreover, to evaluate the level of contamination by Pseudomonas spp. (Centrimide Agar Base), coliforms (Endo Agar) and fungi (Petrifilm YM for yeasts and molds) as well as to identify the bacteria by means biochemical test in form of kit (API 20NE), to detect mouth microorganisms by Checkerboard DNA-DNA Hybridization technique and to analyze the biofilm formed on dental unit waterlines (air-water syringes and high-speed handpieces) by scanning electron microscopy (SEM). The levels os bacteria in water from tap and dental unit were higher by R2A Agar than Petrifilm AC (p<0.001). Although, the tap water used to supply the dental unit reservoirs had few bacteria (908CFU/ml) R2A and (2CFU/ml) Petrifilm AC, water from 25 dental units showed: reservoir from 0 to 3,900,000CFU/ml (average of 211,705FCU/ml) R2A Agar, and from 0 to 610,000 CFU/ml (average of 30,842CFU/ml) Petrifilm AC; air water syringes from 0 to 5,200,000CFU/ml (average of 509,068CFU/ml) R2A Agar, and from 0 to 610,000CFU/ml (average of 30,842CFU/ml) Petrifilm AC; and high-speed handpieces from0 to 6,000,000CFU/ml (average of 862,279CFU/ml) R2A Agar, and from and0 to 730,000CFU/ml (average of 61,817CFU/ml) Petrifilm AC. The Petrifil AC plates incubated at 23ºC for 7 days demonstrated a level of bacterial contamination higher than those at 35ºC for 48h (p<0.001). According to culture method, Escherichia coli, total coliforms and Pseudomonas spp. Were not detected in water from taps, but E. coli and/or total coliforms were presented in 11 (44%) high-speed handpiece water. However, according to molecular method, 1 (50%) water sample from a tap and 36 (48%) from dental units showed contamination with E. coli. Water from 10 taps and 25 dental units were contamined with fungi in 1 (10%) and 17 (68%) samples, respectively. The analysis by SEM showed in all dental unit waterlines biofilms constituted of a microbial diversity embedded in dense and extensive matrices of extracellular polymeric substances. The bacteria identified by API 20 NE were Acinetobacter iwoffiii, Brevundimonas vesicularis, Burkholderia cepacia, Morexella spp., Oligella ureolytica, Pasteurella spp., P. aeruginosa and Sphingomonas paulcimobilis. The oral bacteria prevalent in dental unit water samples were Streptococcus gordonii (35/46.7%), Treponema denticola (28/37.3%) and Aggregatibacter actiomycetemcomitansb (9/25.6%). In conclusion, BIOFILM formed in dental nit waterlines serve as an amplifier system of few microorganisms from tap water, being the major cause of high contamination of dental unit water. Besides, recommendations to maintain the microbiological quality of dental units as well as the fungal evaluation should be employed for Dentistry professionals

O objetivo deste estudo foi avaliar o efeito do hipoclorito de sódio e a clorexidina sobre biofilme dental formado in situcom relação a: concentração do hipoclorito de sódio (1%, 2.5% e 5%) e clorexidina 2%, tempo de exposição à solução irrigadora (5, 15 e 30 minutos.), volumes das soluções (500 µl e 1 mL), espessura do biofilme e área de limpeza segundo analise morfométrica. Foram utilizados 120 blocos de dentina bovina esterilizada, colocados em um aparelho intraoral e utilizados por um voluntário durante 3 dias. Transcorrido o período experimental as amostras foram retiradas e coradas com 50 µl de laranja de acridina para determinar a espessura do biofilme pre irrigação por meio do Microscopio confocal de varredura laser (CLSM). Foram conformados 12 grupos experimentais com 10 blocos cada um e irrigados com NaOCl e clorexina. Dez amostras foram irrigadas com 500 µl (N=5) e 1mL (N=5) de NaOCl 1% por: 5 min (G1), 15 min (G2) e 30 min (G3). Dez amostras foram irrigadas com 500 µl (N=5) e 1mL (N=5) de NaOCl 2.5% por: 5 min (G4), 15 min (G5) e 30 min (G6). Dez amostras foram irrigadas com 500 µl (N=5) e 1mL (N=5) de NaOCl 5% por: 5 min (G7), 15 min (G8) e 30 min (G9). Dez amostras foram irrigadas com 500 l (N=5) e 1mL (N=5) de Clorexidina 2% por: 5 min (G10), 15 min (G11) e 30 min (G12). Para cada sub grupo experimental (N=5) se deixou um sexto bloco o qual foi irrigado com água destilada estéril para procedimentos de controle. Nos grupos de 15 e 30 minutos a solução de NaOCl e clorexidina foi renovada a cada 5 minutos. Os segmentos de dentina foram lavados com 200 µl de água destilada estéril para eliminar resíduos não aderidos e corados com 50 µl de Laranja de acridina para determinar a espessura do biofilme após irrigação por meio do CLSM. Foram encontrados altos valores de dissolução do biofilme e dentina limpa após contato com NaOCl a 5% durante 5 e 15 min. e com todos os grupos de NaOCl durante 30 min. O uso de Clorexidina a 2% não dissolveu o biofilme e nem aumentou a limpeza dentinária quando comparado com o NaOCl (P < 0.05). / Introduction: The aim of this study is to evaluate the biofilm dissolution and cleaning ability of different irrigant solutions on intraorally infected dentin. Methods: 120 bovine dentin specimens were infected intraorally using a removable orthodontic device. 30 samples were used for each irrigant solution; 2% Chlorhexidine, 1%, 2.5% and 5.25% of sodium hypochlorite (NaOCl). The solutions were used for 5, 15 and 30 minutes and 2 experimental volume 500µl and 1mL. The samples were stained using the acridine orange dye before and after the experiments and evaluated using a confocal microscope. The percentage of biofilm, isolated cells and no colonized dentin was measured using a grid system. Differences in the reduction or increase of the studied parameters was assessed using non-parametric methods ( P < 0.05). Results: The higher values of biofilm dissolution and clean dentin were found in the 30 minutes NaOCl groups and in the 5 and 15 minutes of 5.25% NaOCL. The use of 2% chlorhexidine solution does not improve the biofilm dissolution neither increases the cleaning of the dentin in comparison to the NaOCl solutions (P < 0.05). Conclusions: 2% chlorhexidine does not dissolve the biofilms. 30 minutes of sodium hypochlorite are necessary to have the higher values of biofilm dissolution and to increase the cleaning of the dentin independently of the concentration in comparison to the 5 min and 15 min contact time.

Pseudomonas aeruginosa é um patógeno multirresistente capaz de produzir um biofilme protetor contra antibacterianos (ATB). O presente estudo avaliou estratégias terapêuticas contra biofilmes de cepas multirresistentes de P. aeruginosa representativas de clones e/ou genótipos de resistência endêmicos no Brasil. Os biofilmes foram formados in vitro utilizando um modelo adaptado do MBEC Assay e as estratégias terapêuticas utilizaram bacteriófagos líticos, combinação de ATB e/ou uso de força iônica alta (meio FIA). A aplicação de bacteriófagos líticos (&phi;SPM-1) e a combinação de Aztreonam (ATM) e Piperacilina/Tazobactam (PPT), não foram capazes de eliminar o biofilme. Biofilme formado em meio FIA possui CIM similar ao modelo planctônico, tanto para ATM (4 mg/mL) quanto para PPT (16 mg/mL). Ambos os ATB apresentaram CIM reduzida (inferior a 2 mg/mL) quando aplicados em conjunto com meio FIA. Dependendo da concentração de NaCl, a aplicação de meio FIA possui efeito bactericida sobre bactérias planctônicas e efeito bacteriostático sobre biofilmes já formados. / Multidrug-resistant Pseudomonas aeruginosa is a pathogen capable of producing a protective biofilm against antibiotics (ATB). The present study evaluated therapeutic strategies against biofilms of multidrug-resistant strains of P. aeruginosa representative of clones and/or exhibiting resistance genotypes endemic in Brazil. Biofilms were formed in vitro using an adapted model of MBEC Assay and the therapeutic strategies used lytic bacteriophages, combination of ATB and/or use of high ionic strength (HIS medium). The application of lytic bacteriophages (&phi;SPM-1) and the combination of Aztreonam (ATM) and Piperacillin / Tazobactam (PPT) were unable to remove the biofilm. The application of HIS during biofilm formation restored the bacteriostatic effect of both ATM (4 mg/mL) and PPT (16 mg/ml). Both ATB showed reduced MIC values (less than 2 mg/mL) when applied in conjunction with HIS medium. It was shown that HIS has a bacteriostatic or bactericidal effect on planktonic growth, which depend on the NaCl concentration, and bacteriostatic activity against mature biofilm.

Large areas of mesotidal estuaries become subaerial during low tide. Here we study the effect of nutrient enrichment and several meteorological and hydrodynamic parameters on the erodability of mudflat substrates when they are emergent. We tested the impact of nutrient fertilization on tidal flat sediments over a two week period in September 2011 in Plum Island Sound, Massachusetts (USA). High resolution measurements from our experiment indicate that daily nutrient enrichment at 70 μM NO3‾ using our experimental approach does not change the critical shear stress of the muddy substrate, nor affect the concentration of chlorophyll a at the surface. Sediment erodability is instead directly related to the potential evaporation rate and to the duration of the subaerial period. Chlorophyll a concentration decreases when evaporation is high, possibly due to the downward migration of diatoms. Sediment concentrations in the water column during submergence strongly depend on bottom shear stresses triggered by tidal currents. Surprisingly, they are also related to the total evaporation that occurred in the previous emergence period. We conclude that subaerial desiccation at low tide decreases the erodability of mudflat sediments. This strengthening effect is not lost during the following submerged period, thus limiting the erosive effect of tidal currents. For the first time we show that not only subaqueous but also subaerial processes control the erodability of mudflats. Global warming and other climatic variations regulating long-term evaporation rates can therefore directly affect the stability of mudflats in mesotidal environments.

Orientador: Márcia Regina de Moura[UNESP] Aouada / Resumo: O presente trabalho é uma investigação das propriedades de embalagens ativas beneficiadas com a substância Curcuma longa derivada do açafrão. Essa substância apresenta propriedade antioxidativa, que tem despertado interesse da indústria de fármacos, e que vem sendo bastante utilizada na indústria de alimentos como corante natural e tempero. O propósito do trabalho é a síntese e caracterização da solução base para a produção dos filmes ativos contendo composição inédita de Curcuma longa, carboximetilcelulose (CMC) e nanopartículas de quitosana (NSQ). A produção de filmes e revestimentos poliméricos para a indústria alimentícia é alvo de constantes pesquisas, devido a necessidade de diminuir o volume de embalagens plásticas descartadas e otimizar as propriedades e validade dos alimentos. Dentre a imensa variedade de polímeros a carboximetilcelulose é muito favorável a produção de filmes pelo seu caráter atóxico, biodegradabilidade e baixo custo. A quitosana assim como o CMC é uma substância com ampla aplicação nos campos da farmacologia, tecnologia de biomateriais, biomedicina, agricultura e indústrias cosmética e alimentícia. Os filmes foram produzidos com carboximetilcelulose, nanopartículas de quitosana e Curcuma longa de acordo com o método “casting”. As nanopartículas foram obtidas pelo método de gelificação ionotrópica. Foram realizadas análises: térmicas, mecânicas, permeabilidade ao vapor de água, espectroscopia do infra- vermelho, ângulo de contato, microscopia eletrôn... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The present work is an investigation of the properties of active packaging benefited with the substance Curcuma longa derived from saffron. This substance has antioxidative properties, which has aroused interest in the drug industry and is widely used in the food industry as a natural dye and seasoning. The purpose of the work is the synthesis and characterization of the base solution for the production of active films containing novel composition of Curcuma longa, carboxymethylcellulose (CMC) and chitosan nanoparticles (NSQ). The production of films and polymer coatings for the food industry is the subject of constant research, due to the need to reduce the volume of discarded plastic packaging and to optimize the properties and validity of the food. Among the immense variety of polymers the carboxymethylcellulose is very favorable the production of films by its nontoxic character, biodegradability and low cost. Chitosan as well as CMC is a substance with broad application in the fields of pharmacology, biomaterial technology, biomedicine, agriculture and cosmetic and food industries. The films were produced with carboxymethylcellulose, chitosan nanoparticles and Curcuma longa according to the casting method. The nanoparticles were obtained by ionotropic gelation. Thermal, mechanical, water vapor permeability, infrared spectroscopy, contact angle and scanning electron microscopy were performed. The nanoparticles were characterized by zeta potential and presented spherical sh... (Complete abstract click electronic access below) / Mestre

Mes travaux de recherche ont eu pour but d’analyser la diversité des microorganismes des trois domaines du vivant présents dans des biofilms phototrophes exposés à l’air, se développant sur des substrats minéraux divers, afin d’essayer, d’une part, de répondre à des questions de diversité et de biogéographie et, d’autre part, d’étudier le processus de colonisation par le biais d’expériences d’exposition contrôlées.J’ai ainsi caractérisé, essentiellement par des approches moléculaires basées sur l'analyse des banques des gènes d'ARNr de la petite sous-unité (SSU rDNAs) et sur des analyses d'empreintes communautaires, la diversité microbienne (procaryote et eucaryote) formant des biofilms matures (exposés depuis plusieurs années) dans plusieurs sites géographiques en Irlande du Nord, en France et en Ukraine, dans la région de Chernobyl. Dans ces biofilms soumis à forte pression sélective, nous avons mis en évidence beaucoup de microorganismes hétérotrophes et phototrophes, mais avec une diversité relativement restreinte en comparaison à d’autres milieux comme les sols ou les systèmes aquatiques. Les archées étaient absentes. Les conditions environnementales auxquelles ce type de biofilm est constamment exposé comme l’irradiation, la dessiccation et la limitation des nutriments sélectionnent des microorganismes qui développent des stratégies pour s’adapter comme, entre autres, la production de pigments. Ce sont des microorganismes fréquemment retrouvés dans des milieux désertiques extrêmes et résistants aussi aux radiations ionisantes qui ont ainsi été identifiés, notamment des Deinococcales et des Actinobacteria, ou encore des champignons ascomycètes (Ascomycota). Parmi les organismes phototrophes, nous avons dénombré des Cyanobacteria, des algues vertes (Chlorophyta) et des Streptophyta. Nous avons mis en évidence que les facteurs environnementaux influencent la composition des biofilms. Toutefois, tandis que la composition de la communauté bactérienne est fortement dépendante de la nature du substrat ou elle se développe, la composition des communautés microbiennes eucaryotes dépend de la distance géographique. Nous avons également mené des expériences de colonisation en exposant un même substrat minéral dans trois sites géographiques en Irlande du Nord et en France. L'analyse de la diversité microbienne lors du processus de colonisation a révélé des changements importants dans la composition des communautés, que ce soit pour les procaryotes ou pour les eucaryotes avec, cependant, des comportements différents de ces deux groupes de microorganismes. Dans le cas des bactéries, on observe une transition des Gammaproteobacteria, qui dominent les temps 0-6 mois et qui correspondent vraisemblablement aux cellules inactives en dispersion, vers des Betaproteobacteria, Bacteroidetes, Alphaproteobacteria et Actinobacteria dans des phases successives de formation du biofilm. Par contre, dès leur détection sur le substrat minéral, les eucaryotes sont massivement dominés par des champignons ascomycètes et basidiomycètes, des algues vertes ainsi que d'autres composantes minoritaires comme des ciliés, étant détectées dans des stades plus tardifs. Nos résultats montrent que les organismes hétérotrophes sont pionniers dans la formation de ces biofilms, ce qui permet d'émettre l'hypothèse qu'ils facilitent l'installation des cyanobactéries et surtout des algues vertes. Ils montrent aussi que le processus d'assemblage des communautés bactériennes dépend du temps de colonisation, alors que le site géographique détermine celui des microorganismes eucaryotes. Ces différences majeures de comportement pourraient être expliquées par des modes de vie différents entre les organismes de ces deux grands groupes. / The major objective of my PhD work was the analysis of the diversity of microorganisms from the three domains of life associated with phototrophic biofilms developing on different mineral substrates exposed outdoors. These studies aimed at answering questions about microbial diversity and biogeography and also at studying the colonization process through controlled exposure experiments. I have thus characterized, essentially by molecular methods based on small subunit (SSU) rRNA gene libraries and fingerprinting analyses the diversity of prokaryote and eukaryote microorganisms forming mature biofilms (exposed for several years) in various geographic sites in Northern Ireland, France and Ukraine, in the Chernobyl area. In these biofilms, subjected to strong selective pressure, we found many heterotrophic and phototrophic microorganisms, but their diversity was limited when compared to that of other environments such as soils or aquatic systems. Archaea were absent from all biofilms. The environmental conditions to which these biofilms are constantly exposed, such as irradiation, desiccation and nutrient limitation select for organisms that develop particular adaptive strategies including, among others, pigment production. The microorganisms identified in these biofilms are also frequently found in extreme, desert environments and are known for their resistance also to ionizing radiation, such as Deinococcales and Actinobacteria or ascomycete fungi (Ascomycota). Among phototrophic lineages, we identified Cyanobacteria, Chlorophyta (green algae) and sometimes Streptophyta. We showed that environmental parameters influenced biofilm microbial communities. However, whereas the bacterial community composition depends on the nature of the substrate, the microbial eukaryotic community composition depends on the geographic distance. We also carried out colonization experiences exposing outdoors the same mineral substrate in three different sites in Northern Ireland and France. The analysis of microbial diversity along the colonization process revealed important changes in community composition both for prokaryotes and eukaryotes, although the behavior of the two groups was different. In the case of bacteria, we observed a transition from Gammaproteobacteria, which dominated the initial 0-6 months and which likely corresponded to inactive dispersive cells, towards Betaproteobacteria, Bacteroidetes, Alphaproteobacteria and Actinobacteria in successive steps of biofilm formation. By contrast, since their detection on mineral substrates, eukaryotes were massively dominated by ascomycete and basidiomycete fungi, green algae and other minor components such as ciliates were detected in later stages of biofilm formation. Our results show that heterotrophic organisms are pioneers in the formation of these biofilms, leading to the hypothesis that they facilitate the settlement of Cyanobacteria and, especially, of green algae. They also show that the process of bacteria community assembly depends on colonization time whereas the geographic site determines that of eukaryotic microorganisms. These major differences might be explained by different lifestyles between organisms of the two groups